The signaling pathway through which Procyanidin B2 increases the activity of the Na+/K+ ATPase in Caco-2 cells

Procyanidins are plant polyphenolic compounds reported to play a beneficial role in inflammatory bowel disease which is usually accompanied with a reduced activity of the Na+/K+ ATPase. Thus, a correlation between procyanidins and the ATPase was suspected and was examined in this work. The activity of the ATPase was assayed by measuring the amount of inorganic phosphate liberated in presence and absence of ouabain. Caco-2 cells treated with Procyanidin B2 exerted a stimulatory effect on the Na+/K+ ATPase which disappeared in the presence of inhibitors of PI3K, PKC, PKA, and COX enzymes. Exogenous PGE2 stimulated the ATPase via EP4 receptors and PKA. PKC was found to be downstream PI3K and upstream PGE2 while p38MAPK had a modulatory role but was not along the signaling pathway. It was concluded that procyanidin B2 acts via PI3K, activating PKC and leading to PGE2 release. PGE2 then stimulates the ATPase via EP4 and PKA

The epinephrine-induced PGE2 reduces Na+/K+ ATPase activity in Caco-2 cells via PKC, NF-κB and NO.

We showed previously an epinephrine-induced inhibition of the Na+/K+ ATPase in Caco-2 cells mediated via PGE2. This work is an attempt to further elucidate mediators downstream of PGE2 and involved in the observed inhibitory effect. The activity of the Na+/K+ ATPase was assayed by measuring the amount of inorganic phosphate liberated in presence and absence of ouabain, a specific inhibitor of the enzyme. Changes in the protein expression of the Na+/K+ ATPase were investigated by western blot analysis which revealed a significant decrease in the abundance of the ATPase in plasma membranes. Treating the cells with epinephrine or PGE2 in presence of SC19220, a blocker of EP1 receptors abolished completely the effect of the hormone and the prostaglandin while the effect was maintained unaltered in presence of antagonists to all other receptors. Treatment with calphostin C, PTIO, ODQ or KT5823, respective inhibitors of PKC, NO, soluble guanylate cyclase and PKG, abrogated completely the effect of epinephrine and PGE2, suggesting an involvement of these mediators. A significant inhibition of the ATPase was observed when cells were treated with PMA, an activator of PKC or with 8-Br-cGMP, a cell permeable cGMP analogue. PMA did reduce the protein expression of IκB, as shown by western blot analysis, and its effect on the ATPase was not manifested in presence of an inhibitor of NF-κB while that of SNAP, a nitric oxide donor, was not affected. The results infer that NF-κB is downstream PKC and upstream NO. The data support a pathway in which epinephrine induces the production of PGE2 which binds to EP1 receptors and activates PKC and NF-κB leading to NO synthesis. The latter activates soluble guanylate cyclase resulting in cGMP production and activation of PKG which through direct or indirect phosphorylation inhibits the Na+/K+ ATPase by inducing its internalization

Leptin inhibits glucose intestinal absorption via PKC, p38MAPK, PI3K and MEK/ERK.

The role of leptin in controlling food intake and body weight is well recognized, but whether this is achieved by modulating nutrient absorption is still a controversial issue. The aim of this work was to investigate the direct effect of luminal leptin on glucose intestinal absorption and elucidate for the first time its signaling pathway. Fully differentiated Caco-2 cells grown on transwell filters were used for glucose transport studies. Leptin caused a significant reduction in glucose absorption. Individual and simultaneous inhibition of ERK, p38MAPK, PI3K or PKC abrogated completely the inhibitory effect of leptin. Activating PKC, lead to a stimulatory effect that appeared only when ERK, p38MAPK, or PI3K was inactive. Moreover, leptin increased the phosphorylation of ERK, Akt and p38MAPK. This increase changed into a decrease when p38MAPK and PKC were inactivated individually. Inhibiting ERK maintained the leptin-induced up-regulation of p-Akt and p-p38MAPK while inhibiting PI3K reduced the level of p-ERK and p-Akt but maintained the increase in p-p38MAPK. These results suggest that leptin reduces glucose absorption by activating PKC. Although the latter modulates glucose absorption via a stimulatory and an inhibitory pathway, only the latter is involved in leptin's action. Active PKC leads to a sequential activation of p38MAPK, PI3K and ERK which exerts an inhibitory effect on glucose absorption. The results reveal a modulatory role of leptin in nutrient absorption in addition to its known satiety inducing effect

FTY720P inhibits hepatic Na+/K+ ATPase via S1PR2 and PGE2

Sphingosine-1-phosphate (S1P) was found previously to inhibit the Na+/K+ ATPase in HepG2 cells. Whether fingolimod (FTY720), a S1P receptor “(S1PR)” agonist, similarly inhibits the ATPase is a question that needs to be addressed. The aim of this work was to study the effect of FTY720P, the active form of the drug, on the activity of the Na+/K+ ATPase in HepG2 cells and elucidate its mechanism of action. The activity of the ATPase was assayed by measuring the amount of inorganic phosphate liberated in presence and absence of ouabain. FTY720-P (7.5 nM, 15min) reduced significantly the activity of the enzyme. This effect disappeared completely in presence of JTE-013, a specific blocker of sphingosine-1-phosphate receptor 2 (S1PR2) as well as in presence of calphostin and indomethacin, respective inhibitors of Protein kinase C (PKC) and COX-2. The effect of FTY720P was mimicked by prostaglandin E2 (PGE2) and PMA but abrogated by NF-κB inhibition. When NF-κB was inhibited, the effect of exogenous PGE2 still appeared but that of PMA was not manifested suggesting that NF-κB is upstream of PGE2 and downstream of PKC. It was concluded that FTY720P activates via S1PR2, PKC and NF-κB. The latter induces PGE2 generation and the Na+/K+ ATPase inhibition.The accepted manuscript in pdf format is listed with the files at the bottom of this page. The presentation of the authors' names and (or) special characters in the title of the manuscript may differ slightly between what is listed on this page and what is listed in the pdf file of the accepted manuscript; that in the pdf file of the accepted manuscript is what was submitted by the author

PGE2 upregulates the Na+/K+ ATPase in HepG2 cells via EP4 receptors and intracellular calcium.

The Na+/K+ ATPase is a key regulator of the hepatocytes ionic homeostasis, which when altered may lead to many liver disorders. We demonstrated recently, a significant stimulation of the Na+/K+ ATPase in HepG2 cells treated with the S1P analogue FTY 720P, that was mediated through PGE2. The mechanism by which the prostaglandin exerts its effect was not investigated, and is the focus of this work. The type of receptors involved was determined using pharmacological inhibitors, while western blot analysis, fluorescence imaging of GFP-tagged Na+/K+ ATPase, and time-lapse imaging on live cells were used to detect changes in membrane abundance of the Na+/K+ ATPase. The activity of the ATPase was assayed by measuring the amount of inorganic phosphate liberated in the presence and absence of ouabain. The enhanced activity of the ATPase was not observed when EP4 receptors were blocked but still appeared in presence inhibitors of EP1, EP2 and EP3 receptors. The involvement of EP4 was confirmed by the stimulation observed with EP4 agonist. The stimulatory effect of PGE2 did not appear in presence of Rp-cAMP, an inhibitor of PKA, and was imitated by db-cAMP, a PKA activator. Chelating intracellular calcium with BAPTA-AM abrogated the effect of db-cAMP as well as that of PGE2, but PGE2 treatment in a calcium-free PBS medium did not, suggesting an involvement of intracellular calcium, that was confirmed by the results obtained with 2-APB treatment. Live cell imaging showed movement of GFP-Na+/K+ ATPase-positive vesicles to the membrane and increased abundance of the ATPase at the membrane after PGE2 treatment. It was concluded that PGE2 acts via EP4, PKA, and intracellular calcium

The Appearance of a Leptin Effect on Glucose Absorption in Caco2 Cells Depends on Their Differentiation Level

Backdround/Aims: The aim of this work was to study the effect and mechanism of action of leptin added apically, on glucose absorption, using Caco-2 cells as a model. Methods: Cells were grown on inserts and treated with leptin, at different time points after confluence. Radiolabelled glucose was added to the upper chamber and samples from the lower chamber were collected and assayed for radioactivity. Results: Glucose absorption increased with an increase in the level of differentiation and was associated with an increase in the protein expression level of glucose transporters. Leptin reduced glucose absorption only by day 16 after confluence, the time at which apical leptin receptors started appearing. This inhibitory effect became higher the longer the post confluence period, and was prominent on day 23. The hormone effect was found to be mediated via a decrease in the number of glucose transporters (SGLT1 and GLUT2) and a decrease in the activity of the Na+/K+ ATPase which was assayed by measuring the amount of inorganic phosphate liberated in presence and absence of enzyme activators. Conclusion: It was concluded that by day 23 post confluence, Caco-2 cells are differentiated and are appropriate to use as a model for intestinal transport studies

Epinephrine modulates Na+/K+ ATPase activity in Caco-2 cells via Src, p38MAPK, ERK and PGE2.

Epinephrine, a key stress hormone, is known to affect ion transport in the colon. Stress has been associated with alterations in colonic functions leading to changes in water movements manifested as diarrhea or constipation. Colonic water movement is driven by the Na+-gradient created by the Na+/K+-ATPase. Whether epinephrine acts via an effect on the Na+/K+-ATPase hasn't been studied before. The aim of this work was to investigate the effect of epinephrine on the Na+/K+-ATPase and to elucidate the signaling pathway involved using CaCo-2 cells as a model. The activity of the Na+/K+-ATPase was assayed by measuring the amount of inorganic phosphate released in presence and absence of ouabain, a specific inhibitor of the enzyme. Epinephrine, added for 20 minutes, decreased the activity of the Na+/K+-ATPase by around 50%. This effect was found to be mediated by α2 adrenergic receptors as it was fully abolished in the presence of yohimbine an α2-blocker, but persisted in presence of other adrenergic antagonists. Furthermore, treatment with Rp-cAMP, a PKA inhibitor, mimicked epinephrine's negative effect and didn't result in any additional inhibition when both were added simultaneously. Treatment with indomethacin, PP2, SB202190, and PD98059, respective inhibitors of COX enzymes, Src, p38MAPK, and ERK completely abrogated the effect of epinephrine. The effect of epinephrine did not appear also in presence of inhibitors of all four different types of PGE2 receptors. Western blot analysis revealed an epinephrine-induced increase in the phosphorylation of p38 MAPK and ERK that disappeared in presence of respectively PP2 and SB2020190. In addition, an inhibitory effect, similar to that of epinephrine's, was observed upon incubation with PGE2. It was concluded that epinephrine inhibits the Na+/K+-ATPase by the sequential activation of α2 adrenergic receptors, Src, p38MAPK, and ERK leading to PGE2 release

MEK/ERK, p38MAPK, PI3K and PKC act along the same pathway.

<p>Effect of leptin (10 nM) on glucose absorption by Caco-2 cells in presence and absence of (a) Calphostin C + SB202190, inhibitors of PKC and p38MAPK respectively, (b) Calphostin C + Wortmannin, respective inhibitors of PKC and PI3K, (c) PD98059 + Wortmannin, inhibitors of MEK/ERK and PI3K respectively, (d) PD98059 + SB202190 + Wortmannin, respective inhibitors of MEK/ERK, p38MAPK and PI3K. Absorption of glucose was measured at 10-min intervals over a period of 50 min. Values are means ± SEM of 5 observations. * P<0.05, **P<0.01, ***P<0.001: significantly different from all other treatments. </p